Increase In Glutamine Synthetase Activity Research Articles

Correlation of nitrogen metabolism with biosurfactant production by Pseudomonas aeruginosa

A direct relationship between increased glutamine synthetase activity and enhanced biosurfactant production was found in Pseudomonas aeruginosa grown in nitrate and Proteose Peptone media. A chloramphenicol-tolerant strain showed a twofold increase in biosurfactant production and glutamine synthetase activity. Increased ammonium and glutamine concentrations repressed both phenomena.

Different effects of supplied ammonium on glutamine synthetase activity in mustard (Sinapis alba) and pine (Pinus sylvestris) seedlings

The activity of glutamine synthetase (GS) in mustard (Sinapis alba L.) and Scots pine (Pinus sylvestris L.) seedlings was used as an index to evaluate the capacity to cope with excessive ammonium supply. In these 2 species GS activity was differently affected by the application of nitrogen compounds (NH4+ or NO3−). Mustard seedlings older than 5 days showed a considerable increase in GS activity after NH4+ or NO3− application. This response was independent of the energy flux, but GS activity in general was positively affected by light. Endogenous NH4+ did not accumulate greatly after nitrogen supply. In contrast, seedlings of Scots pine accumulated NH4+ in cotyledons and roots and showed no stimulation of GS activity after the application of ammonium. In addition, root growth was drastically reduced. Thus, the pine seedlings seem to have insufficient capacity to assimilate exogenously supplied ammonium. NO3−, however, did not lead to any harmful effects.

Effect of trophic factors, released after hippocampal injury, on astroglial cell proliferation

An increase in astrocyte mitogenic factors and in some specific astroglial enzymatic activities after neuronal injury has been observed. Our study is concerned with the effect of the intracerebral administration of ibotenic acid (IBO) into the rat hippocampus. IBO injection causes a selective degeneration of neurons while sparing afferent fibers. We observed a transient increase in glutamine synthetase activity, a well-known astroglial marker, reaching a peak at 9-15 days after injury in lesioned hippocampus. We investigated the presence of astrocyte mitogenic factors at various times after toxin injection. Crude extracts, prepared from lesioned hippocampi 4, 9, and 14 days after IBO injection, were tested for the ability to stimulate [methyl-3H]thymidine incorporation into rat astroglial cell cultures. Crude extracts prepared 9 and 14 days after IBO injection showed a higher mitogenic activity compared to extracts prepared 4 days after lesion. Mitogenic activity of injured brain extracts was suppressed by heat inactivation (100 degrees C for 10 min).

Effects of Budesonide and Dexamethasone on Cell Morphology, Thymidine Incorporation and Glutamine Synthetase Activity in Rat Primary Astroglial Culture

Glucocorticoids are widely used in medical practice. Since astrocytes are target cells for glucocorticoids, the effects of two glucocorticoids, budesonide and dexamethasone, on cell morphology, thymidine incorporation into DNA and glutamine synthetase (GS) activity, were evaluated in primary astroglial cultures. Both budesonide and dexamethasone caused a threefold increase in GS activity over a dose range of 10-6–10-9 M. There were no differences between the effects of the two glucocorticoids. No morphological changes were obtained with the glucocorticoids with the same concentration range as above and with incubation periods of up to 48 hours. Nor were any effects on [3H]-thymidine incorporation observed. It is concluded that glucocorticoids can regulate intracellular amino acid metabolism in astrocytes by an activation of GS, while cell morphology and cell replication appear not to be affected.

Ethanol in Clinically Relevant Concentrations Enhances Expression of Oligodendroglial Differentiation but Has No Effect on Astrocytic Differentiation or DNA Synthesis in Primary Cultures

Primary cultures of cerebral glia derived from newborn rat brain were utilized to evaluate the effects of ethanol on DNA synthesis and cellular differentiation. Glutamine synthetase was employed as a marker for astrocytic differentiation and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP), for oligodendroglial differentiation. Concentrations of ethanol of 17-86 mM, i.e., a concentration range comparable to that observed in humans, were utilized. No effect of ethanol on DNA synthesis was observed, despite the use of synchronized cultures. Similarly, no effect of ethanol on the developmental increase of glutamine synthetase activity was seen, despite the use of astrocytes purified by the selective detachment technique and a prolonged duration of exposure to ethanol (17 days). In contrast, however, a striking enhancement of CNP activity was demonstrable in both mixed glial cultures and in oligodendroglia purified by the selective detachment technique. In the latter cells, the stimulatory effect of ethanol was evident within 9 days of exposure, and after 17 days of exposure, ethanol-treated cells exhibited a two-fold higher CNP activity than did control cells. This peak effect was observed at an ethanol concentration of only 17 mM. Thus, these data indicate that (1) clinically relevant concentrations of ethanol have no effect on either DNA synthesis or on at least one expression of astrocytic differentiation in glial primary cultures, but (2) these concentrations exert a striking enhancement of CNP activity, a marker of oligodendroglial differentiation. The findings have implications both for the effects of ethanol on the developing nervous system and the regulation of oligodendroglial differentiation.

Dexamethasone regulates glutamine synthetase expression in rat skeletal muscles.

The regulation of glutamine synthetase expression by dexamethasone was studied in rat skeletal muscles. Daily administration of dexamethasone caused striking enhancement of glutamine synthetase activity in plantaris, soleus, and diaphragm muscles. Northern blot analysis revealed that the dexamethasone-mediated increase of glutamine synthetase activity was associated with dramatically increased levels of glutamine synthetase mRNA. Both glutamine synthetase activity and mRNA levels were significantly elevated in plantaris muscle at 0.5 mg.kg-1.day-1 of dexamethasone, a dose that approximates endogenous corticosteroid levels in animals under severe stress. Quantification of changes in glutamine synthetase mRNA on the basis of total mRNA (by oligo dT hybridization) also revealed a major increase in glutamine synthetase mRNA. Dexamethasone was without effect on beta-tubulin mRNA levels, indicating that glutamine synthetase induction is not part of a global response to glucocorticoids. Dexamethasone treatment resulted in only an approximately 15% increase in glutamine synthetase activity in heart; there was no change in glutamine synthetase mRNA level in this tissue. Thus glucocorticoids regulate glutamine synthetase gene expression in rat skeletal muscles.

Glutamine metabolism in rat skeletal muscle wounded with lambda-carrageenan.

Wounding with lambda-carrageenan results in a marked decrease in the intracellular-free glutamine content of rat skeletal muscle. The potential mechanisms for this finding, including alterations in glutamine release, glutamine utilization, and glutamine synthesis, were investigated in rats under pentobarbital anesthesia. Wounding did not increase glutamine release from muscle during incubation or isolated hindlimb perfusion. Wounded muscle utilized more glutamine than nonwounded muscle, as measured both by the production of [14C]O2 and of -glutamate from labeled glutamine. Maximal glutamine synthetase activity was increased by wounding. The increase in glutamine synthetase activity in wounded muscle was prevented by adrenalectomy and restored by replacement doses of corticosterone in wounded adrenalectomized animals. The decrease in muscle free glutamine induced by wounding is therefore not mediated by an increase in the release of this amino acid, nor by a reduction in the tissue capacity for glutamine synthesis, but by an increase in glutamine utilization at the site of injury. This difference is apparently determined by the utilization of glutamine by the cellular components of the inflammatory infiltrate, which were shown to be capable of active glutaminolysis.

Effect of removal of glutamine and addition of dexamethasone on the activities of glutamine synthetase, ornithine decarboxylase and lactate dehydrogenase in primary cultures of forebrain and cerebellar astrocytes

The regulation of glutamine synthetase (GS) and ornithine decarboxylase (ODC) was studied in primary cultures of two types of astrocytes derived from either newborn forebrain or 8-day-old cerebellum of the rat. In the 14-day-old cultures the specific activities of both these enzymes were about twice as great in forebrain astrocytes as in cerebellar astrocytes. Treatment with dexamethasone or removal of glutamine from the culture medium caused a marked increase in the specific activity of GS. The glutamine-mediated relative increase in GS activity was similar in both types of astrocytes. Removal of glutamine caused a transient reduction in ODC activity in the forebrain astrocytes, while in cerebellar astrocytes the activity remained markedly decreased throughout the period of glutamine deprivation. The severe reduction in ODC activity had relatively little effect on the cell numbers or protein content of the astrocyte cultures. The increase in GS activities, involving protein synthesis de novo, caused by removal of glutamine and by addition of dexamethasone, were additive and therefore probably mediated by different mechanisms. The induction of GS after glutamine removal was blocked by cycloheximide but not by α-amanitin, suggesting regulation at the post-transcriptional level. In contrast, the dexamethasone-mediated induction of GS appeared to be regulated at the transcriptional level, as it was markedly reduced by α-amanitin. None of these conditions had any effect on lactate dehydrogenase activity. Treatment with α-amanitin resulted in a complete suppression of the activity of ODC (a protein with a very short half life), in both the control and dexamethasone treated cultures. However, this enzyme activity was reduced only partially in astrocytes cultured in glutamine deficient medium, suggesting that under these experimental conditions the mRNA may be markedly stabilized in astroglial cells.

Responses in astrocytic C6 glioma cells to ethanol and dibutyryl cyclic AMP

Late passage C-6 glioma cells exhibit astrocytic properties as shown by a characteristic cell morphology and by high levels of the astrocytic cell marker glutamine synthetase (GS). In this study the effects of ethanol (0.2%–1.0% w/v) on the pattern of dibutyryl cyclic AMP (dBcAMP, 1 mM) -induced differentiation were examined using cell number and DNA content as indices for proliferation and cell morphology and GS activity to evaluate differentiation. Differences were observed in the susceptibility of cells to dBcAMP alone, ethanol alone, or simultaneous exposure to both drugs, when cultures were compared at logarithmic and postconfluent phases of growth. Exposure to dBcAMP decreased cell proliferation, induced a characteristic change in cell shape and increased GS activity. In logarithmic phase, simultaneous exposure of cells to ethanol and dBcAMP delayed the dBcAMP-induced change in cell shape and attenuated the mitosis-restricting properties of exposure to dBcAMP. Furthermore, GS activity was greater in dually treated cultures than in cultures treated with dBcAMP alone. We interprete this higher enzyme activity to be the consequence of increased cell-cell contact resulting from larger numbers of cells in the dually treated cultures, coupled with a subsequent dBcAMP-induction of this cytosolic enzyme. In postconfluent cultures, ethanol-exposure did not statistically alter DNA content; whereas GS activity was lower, suggesting that synthesis of GS may be impaired by cellular exposure to ethanol. Furthermore, enzyme activity was also lower in cultures treated with dBcAMP in concert with ethanol than in those treated with dBcAMP alone. These findings indicated that cellular differentiation, as manifested by the dBcAMP-induced changes in cell morphology may be vulnerable to ethanol toxicity. Additionally, the differences between logarithmic and postconfluent phase cultures in GS activity following exposure to ethanol and dBcAMP indicate that the growth kinetics of cultures influence the susceptibility of cells to perturabation by ethanol.

Triiodothyronine increases glutamine synthetase activity in primary cultures of rat cerebellum.

Dissociated cells from 2-3 day-old rat cerebella were cultured in absence of thyroid hormones using conditions yielding mainly glial cells. After 7, 14 and 21 days in vitro, triiodothyronine (60 nM) was added to a set of dishes and glutamine synthetase activity was measured after 24, 48, and 72 h in both control and triiodothyronine-treated cultures. Basal glutamine synthetase activity increased more than 6 X between 7 and 21 days of culture. Triiodothyronine produced significant increases of glutamine synthetase activity after 72 h in 7-day-old cultures (+ 16%), after 48 h in 14-day-old cultures (+ 45%) and after 24 h in 21-day-old cultures (+ 27%). This effect depends on the initial plating density and is not observed if cells are plated at less than 1 cerebellum equivalent per 60 mm dish. Dose-response experiments indicated that 10(-8) M of triiodothyronine induces maximal response whereas half-maximal response is achieved around 10(-10) M. These results show that physiological amounts of thyroid hormone can influence the maturation of astrocytes in culture.

Effect of dexamethasone on glutamine synthetase and glial fibrillary acidic protein in normal and transformed astrocytes.

The effect of dexamethasone on astrocyte differentiation was investigated in vitro, using cultures of normal and transformed astrocytes. The astrocyte-enriched proteins glutamine synthetase (GS) and glial fibrillary acidic (GFA) protein were used as markers of astrocyte differentiation. Ethanol, the vehicle for dexamethasone, decreases GS activity and increases GFA protein concentration in cultures of the established cell line U251MG and in the majority of cultures of transformed astrocytes derived from varying grades of astrocytoma. Ethanol has no effect on primary cultures of astrocytes derived from immature rat brain. Dexamethasone increases GS activity and decreases GFA protein concentration in cultures of U251MG and a grade IV astrocytoma-derived culture, in comparison with ethanol control. Our results show differential effects of two factors on cell-specific proteins in normal and transformed astrocytes.

Regulation of glutamine synthetase and glutaminase activities in cultured skeletal muscle cells.

Glutamine is synthesized in skeletal muscle, released to the circulation, and transported to other tissues, where it may provide important substrate for gluconeogenesis, ammoniagenesis, and energy-yielding pathways. With the ultimate goal of delineating the factors that control glutamine production and release by skeletal muscle, we have studied the regulation of two key enzymes, glutamine synthetase and glutaminase, in the L6 line of rat skeletal muscle cells grown in monolayer culture. The cultured myotubes were found to have glutamine synthetase and phosphate-dependent glutaminase activities. Glutamine synthetase activity was increased following incubation (1) in glutamine-free medium (threefold); (2) in medium containing high glutamic acid concentrations (fourfold); and (3) in medium supplemented with dexamethasone (threefold). In each case the increase in glutamine synthetase activity required several hours to reach a maximum and was prevented by cycloheximide, suggesting that the change occurred through increased enzyme biosynthesis. No substances tested were found to affect glutaminase activity. We conclude that glutamine synthetase in cultured skeletal muscle is responsive to substrate, product, and hormonal regulation.

Relation of cholesterol to astrocytic differentiation in C-6 glial cells.

The relation of cellular cholesterol content to a biochemical expression of astrocytic differentiation was investigated in cultured C-6 glial cells. The astrocytic marker, glutamine synthetase, was studied. Cellular sterol content was perturbed with compactin, a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase and, thereby, cholesterol biosynthesis. Depletion of cellular sterol resulted in 72 h in a more than twofold increase in glutamine synthetase activity. Production of various degrees of sterol depletion with different concentrations of compactin demonstrated a striking inverse relationship between glutamine synthetase activity and the cellular sterol/phospholipid molar ratio. That the effect of compactin, in fact, is mediated by depletion of sterol was shown further by prevention of the compactin-induced increase in synthetase activity by simultaneous addition of exogenous cholesterol. Moreover, addition of cholesterol alone to the culture medium led to both a decrease in glutamine synthetase activity and an increase in the sterol/phospholipid molar ratio. The possibility that the compactin-induced increase in glutamine synthetase activity is caused by an increase in synthesis of the enzyme was suggested by prevention of the increase by cycloheximide. The data suggest that astrocytic differentiation is stimulated by a decrease in cellular sterol content. When considered with our previous observation that oligodendroglial differentiation is inhibited by such a decrease, the findings suggest that cellular sterol content is a critical determinant of the direction of glial differentiation, i.e., whether along astrocytic or oligodendroglial lines.

Glial cells dissociated from newborn and aged mouse brain.

Changes occurring with days in culture and cell passage in cultured glial cells derived from newborn vs aged (18-mo) mouse cerebral hemispheres were compared. The activities of the enzymes glutamine synthetase (GS), an astrocyte marker, and 2',3'-cyclic nucleotide 3'- phosphohydrolase (CNP), an oligodendrocyte marker, were determined. In addition, glial fibrillary acidic protein (GFA) and glycerol phosphate dehydrogenase (GPDH) immunoreactivity was used to morphologically identify astrocytes and oligodendrocytes, respectively. In cultures derived from newborn mouse cerebral hemispheres, both GS and CNP activity and GFA-positive and GPDH-rhodamine-positive cells were present with cell passage. In general, GS activity did not change in early cell passage in cultures from either newborn or aged mouse; in passage 5, GS was high in both sources of cell populations. CNP activity increased with cell passage in cultures derived from newborn mouse; in cultures derived from aged mouse CNP was low in the primary cultures, increased with cell passages 2 and 3, and declined with passages 4 and 5. The survival of astrocytes as shown by GS and the decline in oligodendrocytes as shown by CNP was also supported by an increase in the proportions of GFA and GPDH immunoreactive cells. We interpret the increase in GS activity to parallel the astrogliosis observed in vivo in the aging brain. Moreover, the decline in oligodendrocytes in culture may represent a shift of balance between glial cell types that appears to be influenced by the age of brain tissue and time in culture.

Phytochrome-Mediated Increase in Glutamine Synthetase Activity in Photosensitive New York Lettuce Seeds

Phytochrome-Mediated Increase in Glutamine Synthetase Activity in Photosensitive New York Lettuce Seeds

Epidermal growth factor (EGF) stimulation of cultured brain cells. I. Enhancement of the developmental increase in glial enzymatic activity

Serum-free aggregating cell cultures of fetal rat telencephalon grown in the presence of 3 ng/ml (5 × 10 −10 M) epidermal growth factor (EGF) until day 12 showed 2- to 3-fold increased activities in the two glial enzymes, glutamine synthetase (GLU-S) and 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNPase). This effect was concentration-dependent, with maximal stimulation in cultures treated daily with 3 ng/ml EGF. Addition of EGF during the first 10 culture days was sufficient to produce a maximal stimulation of both GLU-S and CNPase on day 19, whereas treatments starting on day 12 were ineffective. The stimulation of GLU-S preceded that of CNPase. The EGF-induced increase in GLU-S activity was not directly dependent on the presence of insulin, triiodothyronine, or hydrocortisone in the medium, whereas insulin was required for the stimulation of CNPase. A single dose of 5 ng/ml EGF on day 2 caused a slight but significant decrease in DNA synthesis after day 6. The present results indicate that in serum-free aggregating cell cultures of fetal rat telencephalon EGF partially inhibits DNA synthesis, and stimulates an early step in glial differentiation.

Glutamate and glutamine metabolism in tissues of developing lambs

SUMMARYThe activities of glutamine synthetase, phosphate-dependent glutaminase, phosphate-independent glutaminase, glutamate dehydrogenase, γ-glutamyl transferase and glutamine-oxo-acid aminotransferase were assayed in liver, kidney cortex, brain (cerebral hemispheres), spleen, skeletal muscle and ileum obtained from lambs of 100–260 days conceptual age. A curve was fitted to each set of data relating enzyme activity and conceptual age.In the ileum, glutaminase and γ-glutamyl transferase activities declined during development. Glutamine synthetase activity in the spleen increased markedly after birth, whereas glutamate dehydrogenase activity declined as rumen function was established. In the liver, glutamate dehydrogenase and glutamine synthetase activities were highest in suckling lambs and there was a gradual increase in hepatic γ-glutamyl transferase activity throughout the period studied. The activity of phosphate-dependent glutaminase was lowest in the kidney cortex of ruminating lambs but renal activities of glutamate dehydrogenase, phosphate-independent glutaminase, glutamine synthetase and γ-glutamyl transferase were highest in ruminating lambs. In skeletal muscle, a gradual increase in glutamine synthetase activity occurred after 180 days conceptual age, whereas there was no detectable glutaminase activity in ruminating lambs. In the brain, there was an increase in glutamate dehydrogenase, phosphatedependent glutaminase and glutamine synthetase activities during the foetal and early suckling periods, whereas γ-glutamyl transferase activity increased throughout the period studied.Glutamine-oxo-acid aminotransferase activity was not detected in any of the tissues studied. Phosphate-independent glutaminase activity was always less than 10% of phosphate-dependent glutaminase activity and therefore must have a minor role in the metabolism of glutamine in lambs.A consideration of the relative activities of the enzymes at different stages of development indicated that the ileum, spleen, liver, kidney cortex and brain have a substantial potential for glutamine utilization during foetal life. As a lamb matures after birth, there are changes in the metabolism of glutamate and glutamine which indicate that there is a greater potential for net glutamine synthesis in older lambs. This could be associated with the need for detoxification of ammonia in ruminating lambs.

Regulation of in vivo glutamine synthetase activity by glucocorticoids in the developing rat brain

Induction of glutamine synthetase in vivo by glucocorticoids was studied in different brain regions of the rat during development. Corticosterone treatment resulted in an age-dependent increase in glutamine synthetase activity. In 11-day-old rats, in comparison with controls, the increase was about 80% in the cerebellum, 50% in the olfactory bulbs and 20% in the forebrain. During development the effect diminished markedly and at day 20 a marked increase was detectable only in the cerebellum, suggesting that the elevation of glutamine synthetase was dependent on the maturational state of the region at the time of hormone treatment. The increase in enzyme activity was dose-dependent, and was also observed after treatment with dexamethasone but not with testosterone, estradiol or progesterone. Pre-treatment with the protein synthesis inhibitor anisomycin blocked the hormonal response. Also, immunochemical detection of glutamine synthetase in brain homogenates fractionated by SDS-polyacrylamide gel electrophoresis and transferred to diazobenzyloxymethyl paper showed that the increase in enzyme activity was due to induction of protein synthesis. This may be the first report showing a regulation by glucocorticoids of mammalian brain glutamine synthetase in vivo, and it is in good agreement with previous findings with chick retina in vivo and various avian and mammalian cells in vitro.

Glutamine synthetase from the plant fraction of Phaseolus root nodules : Purification of the mRNA and in vitro synthesis of the enzyme

Antiserum raised to glutamine synthetase (GS) from the plant fraction of Phaseolus vulgaris root nodules was used to immunoprecipitate in vitro translation products of nodule and root poly(A) + RNA. The results showed that the large increase in GS activity which occurs during nodulation is due to an increased supply of GS mRNA. A 3-fold enrichment of GS mRNA was obtained by centrifugation of total nodule poly. (A) + RNA on dimethylsulphoxide-formamide-containing sucrose density gradients and purification to near homogeneity was achieved by immunopurification of nodule polysomes.

Four isoforms of glutamine synthetase in light-grown soybeans

Glutamine synthetase (GS) activity recovered from linear sucrose gradients was associated with the cytosol of cells isolated from etiolated soybean hypocotyls whereas light-grown tissue contained increased GS activity localized in both the cytosol and chloroplasts. DEAE-cellulose chromatography indicated two GS isoforms in etiolated hypocotyls whereas light-grown hypocotyls and primary leaves contained four isoforms. Only one GS isoform was recovered from both etiolated and light-grown cotyledons.